Carbohydrates are biologically important but synthetically challenging biomolecules. Fluorous-tagged glycans with an oligo(ethylene glycol) linker are well tolerated glycosyltransferase substrates for high-yield one-pot multienzyme (OPME) synthesis and facile fluorous solid-phase extraction (FSPE) purification of glycans.
Oligo(ethylene glycol)-linked light fluorous tags have been found to be optimal for conjugating to glycans for both high-yield enzymatic glycosylation reactions using OPME systems and quick product purification by FSPE cartridges. The combination of OPME glycosylation systems and the FSPE cartridge purification scheme provides a highly effective strategy for facile synthesis and purification of glycans.
Tagging organic compounds with a light fluorous tail such as a perfluorooctyl (C8F17) or perfluorohexyl (C6F13) group followed by product purification using fluorous solid-phase extraction (FSPE) has found increasing synthetic uses. For carbohydrate synthesis, non-cleavable single, double, and cleavable fluorous tags have been used in the acceptor glycans to allow solution-phase synthesis and fast product purification. Light fluorous protecting groups have also been used in solution-phase or solid-phase synthesis. Odorless fluorinated thioglycosyl donors were prepared and shown excellent reactivities in glycosylation reactions. Mono-perfluoroalkyl (e.g. C8F17- and C6F13) tags and a di-C6F13-tag allowed non-covalent immobilization of fluorous-tagged monosaccharides and oligosaccharides on fluorocarbon-coated glass slides to generate glycan microarrays that can sustain washing processes in carbohydrate-lectin binding studies.
Except for a limited number of examples (e.g. feeding cultured animal cells with fluorous-tagged lactosides or an N-acetylglucosaminide for producing small amounts of elongated fluorous oligosaccharides, using a C3F7 tag to facilitate the product purification of enzymatically synthesized heparosan oligosaccharide derivatives, and using non-covalently immobilized light fluorous tagged glycans as substrates for glycosyltransferase and glycosidase activity assays by mass spectrometry), light fluorous tags have not been used broadly to facilitate product purification in preparative-scale enzymatic synthesis.
Without being bound by theory, this is most likely due to either the lower tolerance of relatively long fluorous tags (e.g. a C8F17 or longer tag) by enzymatic reactions which often leads to no reaction or low yields, or the lower efficiency in FSPE cartridge-purification inherited by very short fluorous tags (e.g. a C3F7 tag).
Accordingly, there is a need to develop OPME systems that tolerate long fluorous tags, which would enable FSPE on a preparative scale. The present disclosure provides a solution to this need.